The Molecular Mechanism Of Lung Adenocarcinoma Cell Death And Vascular Endothelial Cell Apoptosis By Use Of Novel Small Chemical Molecules

OBJECTIVE AND SIGNIFICANCELung cancer mortality stands the first within all kinds of cancer.Among all types of lung cancer,lung adenocarcinoma occurs most commonly and its incident rate is the highest.Lung adenocarcinoma is insensitive to radiation therapy,and it is taboo against operation after metastasis.Therefore,chemotherapy is usually selected to cure lung adenocarcinoma.Current challenges in lung adenocarcinoma treatment include the search of the most promising new agents,which can be integrated into current methods of treatment,and clarification of the mechanism by which lung adenocarcinoma cells undergo growth,proliferation,differentiation,metastasis and death.However,few researches focused on lung adenocarcinoma cell autophagic death and the mechanism of it is poorly understood.Therefore,the further study on the mechanism of autophagic death in lung adenocarcinoma cells is of important theoretical significance and practical value.Why A549 cells were used to explore the molecular mechanisms of autophagy for lung adenocarcinoma in this study? First,A549 cells belong to lung adenocarcinoma cell lines.Second,in the field of anti-lung carcinoma drug discovery,A549 cells are often used as a model for cell-based assays.Third,at present,only a few fragmentary works has been done to explore the molecules involved in A549 cell autophagy and the mechanism of its autophagic death is still unclear.Chemical genetics is a powerful tool for the researches on signal transduction,protein function and discovering new drugs.Several proteins/genes, which play key roles in some biological process,can be identified from the perspective of chemical genetics.It is helpful to illuminate the mechanism of some complex intracellular phenomena.Therefore,chemical genetics provides a new strategy for studying the underlying molecule mechanism of cell death.Based on these background,small chemical molecules were used as a powerful tools to interfere with intracellular signal transduction networks related to cell proliferation, and to systematically investigated the mechanisms of autophagic death in A549 cells. The results of this study not only promote our understanding on the molecular mechanisms of autophagic death of A549 cells,but also provide theoretical bases for developing anti-lung adenocarcinoma agents to a certain extent.Angiogenesis is very important for tumor cell growth and progression. Therefore,inhibiting the proliferation of cancer cell by preventing angiogenesis is an effective way for cancer therapy.Some studies showed that activation and proliferation of vascular endothelial cells(VEC)play important roles in angiogenesis. Apoptosis of vascular endothelial cells would result in angiogenesis degeneration for tumor.Human umbilical vascular endothelial cell(HUVEC)is an excellent model for research on VEC apoptosis.At present,in the studies of HUVEC apoptosis,cell apoptosis generally was induced by depriving of growth factor with or without apoptosis inducer.However,in the presence of growth factor,the molecular mechanism of HUVEC apoptosis is still unclear.In this study,we used small chemical molecules as HUVEC apoptosis inducer to investigate the molecule mechanism of apoptosis in the presence of FGF 2.These data would promote our understanding on HUVEC apoptosis and offered the valuable information for further study in the degeneration of tumor vascular,which might provide ideas and objects for lung adenocarcinoma therapy.STUDY CONTENTS1.The apoptosis of A549 cell were induced by three novel morpholin-3-one derivatives and the potential roles of P53 and Fas proteins in A549 cells apoptosis were studied.2.The effects of benzoxazine derivatives on A549 cell viability were measured by MTT assay,and chemical molecules,which significantly inhibited the growth of A549 cells,were acquired including benzoxazine derivative compound 3f (DBO).3.As a most effective growth inhibitor of A549 cells among benzoxazine derivatives,the effect of DBO on autophagic death ofA549 cells was analyzed.4.In our study above,we found that integrinβ4 was obviously down regulated in A549 cells treated with DBO.Therefore.We further investigated the action of integrinβ4 in autophagic death of A549 cells.5.In the presence of FGF 2.the molecular mechanism of HUVEC apoptosis induced by DBO and the effect of DBO on angiogenesis were analyzed.METHODS1.Measurement of cell death mode:1.1 The changes of cell morphology were observed under phase contrast microscope.1.2 The cell viability was determined by MTT assay to screen small molecules1.3 Nuclear fragmentation,and the quantity and intracellular distribution of acidic vesicle were analyzed by acridine orange(AO)staining under fluorescent microscope.Accordingly,we estimated cell death mode:apoptosis or autophagic death?1.4 To estimate whether autophagic cell death was induced,the content and intracellualr distribution of MAP1 LC3-Ⅱ(LC3-Ⅱ)protein were detected by immunocytochemistry.1.5 To estimate whether necrosis happened,the activity of lactate dehydrogenase in culture medium was measured.1.6 Cell apoptotic rate was detected by TUNEL assay.1.7 The viability of A549 cell was examined by trypan blue exclusion.1.8 The distribution of cell cycle was anlyzed by flow cytometry.2.Analyses of protein contents and distribution:2.1 The contents and intracellular distribution of P53,Fas,integrinβ4 and MAP1 LC3-Ⅱproteins were examinated by immunocytochemistry.2.2 The contents of P53 and integrinβ4 proteins were analyzed by western blot assay.3.Effects of DBO on genes expression in A549 cells:The analysis of gene expression was performed by 22K human genome cDNA microarray according to Patterson et al[Patterson et al.,2006].4.RNA expression analyses of RIS1,FST,RIN2,RAB5B and integrinβ4: The mRNA abundances of RIS1(ras induced senescence 1),FST(Follistatin), RIN2(Ras and Rab interactor 2),RAB5B and integrinβ4 were analyzed by semi-quantitative RT-PCR and agarose gel electrophoresis.5.RNAi:When the contents of integrinβ4 in cells was down-regulated by RNAi,the cell viability was examined by MTT assay and cell death mode was identified by TUNEL assay,LDH activity assay,AO staining and LC3-Ⅱimmunocytochemistry.6.HUVEC culture:The extraction and culture of HUVEC were carried out according to Jaffe et al. [Jaffe et al.,1973].7.Measurement of ROS levelROS level was analyzed by the fluorescent probe(DCHF).8.Activity assay of SODSOD activity was performed according to the protocol of SOD detection kit.9.Activity measurement of NADPH oxidaseActivity of NADPH oxidase was detected according to Li et al.[Li et al.,2002].10.The models of angiogenesis in vitro and in vivo10.1 Capillary-like tube formation assay:The formation of vascular-like structures in HUVECs was analyzed by growth-factor-reduced Matrigel according to Kureishi et al.[Kureishi et al.,2000].10.2 Chick embryo chorioallantoic membrane(CAM)assay:CAM assay was performed as described previously[Zhao et al,2005].RESULTS:1.The molecular mechanisms of A549 cell apoptosis induced by novel morpholin-3-one derivatives1.1 Three novel morpholin-3-one derivatives(compound 1,2 and 3,figure 1) significantly inhibited A549 cell viability in a dose-dependent manner at 48 h (figure 2).1.2 After the cells were treated with 40μg/ml compound 1 for 48 h,the percentage of the cells in S phase obviously decreased and that in G1 and G2/M phases increased,compared to control.After the cells were incubated with 40μg/ml compound 2 and 3 for 48 h,the percentage of the cells in S and G2/M phases obviously decreased,and that in G1 phase obviously increased compared to control(figure 3).1.3 The changes of morphology associated with apoptosis occurred when the cells were treated with 40μg/ml morpholin-3-one derivatives for 48 h(figure 4),for example cell shrink,increasing of intracellular vacuoles,nuclear condensation, weaker refraction and apoptotic body formation.1.4 Treated with 40μg/ml three morpholin-3-one derivatives for 48 h,the cells presented chromatin condensation and DNA fragmentation by AO staining. Among the morpholin-3-one derivatives,compound 3 was the most effective apoptosis inducer(figure 5).1.5 When the cells were separately treated with 40μg/ml morpholin-3-one derivatives(compound 1,2 and 3)for 48 h,P53 protein was accumulated in nucleus and its relative content was significantly increased compared to control (figure 6).The relative content of Fas protein was also remarkably increased and its distribution on cell membrane was altered in the cells treated with the morpholin-3-one derivatives compared to control(figure 7).In the control group, Fas was diffusely distributed on the cell surface.However,in the cells treated with morpholin-3-one derivatives,Fas was assembled into patches and some patches were found to migrate toward one pole of the cell(figure 7).The results are similar with these of our previous report that safrole oxide could induce A549 cell apoptosis through Fas signal pathway.Therefore,we did not further explore the molecular mechanisms of A549 cell apoptosis induced by morpholin-3-one derivatives.2.The primary screen of benzoxazine derivativesAt 24-48 h,200 or 400μM compounds 3c,3d and 3f obviously inhibited A549 cell growth(figure 9),but the compounds 3a.3b,3e and 3g had no obvious effects on it(figure 10).Among compounds 3c,3d and 3f,compound 3f (6,8-dichioro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine,DBO)was the most effective one.3.The mechanism of A549 cell autophagic death induced by DBO3.1 Incubation with 200μM DBO for 48 h resulted in a substantial G0/G1 and G2/M1 phase block in A549 cells(figure 12).The level of P53 protein in cells treated with DBO was higher than that in control group(p＜0.05,figure 13).The relative content of integrinβ4 decreased obviously(p＜0.05,figure 14).These data suggested that DBO inhibited A549 cell viability probably through decreasing the level of integrinβ4,elevating the level of P53 and blocking A549 cells at G1 phase.3.2 TUNEL assay and LDH release analysis showed that there was no significant difference in cell apoptotic rate and LDH activity present in the medium of both control and test groups(figure 16 and 17).A549 cells showed quite extensive acridine orange staining,which is one of the indicatives of autophagic cell death (figure 18).DBO elevated the level of LC3-Ⅱ,which is now widely used as a specific marker of autopgagic cell death,and fluorescent dots were observed in DBO-treated A549 cells,while LC3-Ⅱwas diffusely distributed in the cytoplasm in control group(figure 19).These data suggested that DBO induced autophagic cell death instead of apoptosis and necrosis in A549 cells.3.3 The results from gene microarray showed that 77 genes were differentlv expressed more than two folds in A549 cells treated with 200μM DBO for 48 h (figure 20),including 64(0.3%)up-regulated genes and 13(0.06%) down-regulated genes.Based on the changes of cell growth and the increment of acidic vesicle,we selected RIS1,FST,RAB5B and RIN2 as candidates to be further investigated.DBO gradually increased the expressions of FST,RIS1 and RIN2 and decreased the expression of RAB5B with prolonged treatment(figure 21).It is interesting that the expression of integrinβ4 was recovered at 24 h, compared to control,indicating that this integrin subunit might change with cell cycle.To demonstrate the role of integrinβ4 in controlling A549 cell growth,we used integrinβ4 specific siRNA to down-regulate it.When the expression of integrinβ4 was blocked by the 40 nM siRNA treatment for 48 h.the cell viability decreased significantly(figure 22),while neither apoptosis nor necrosis happened(figure 23).Interestingly,in the cells treated with integrinβ4 specific siRNA,acidic vesicles accumulated and dispersed in cytoplasm(figure 24).In line with the results of AO staining,the LC3-Ⅱaggregated in A549 cells treated with integrinβ4 specific siRNA and the level of LC3-Ⅱalso increased compared to control(figure 24).Moreover,A549 cells treated with integrinβ4 specific siRNA showed significantly increased expressions of RIS1,FST and RIN2,and decreased expression of RAB5B(figure 25).These data indicated integrinβ4, FST,RIS1.RAB5B and RIN2 might were involved in regulating A549 cell growth and autophagic cell death.4.The apoptosis mechanism of HUVEC induced by DBO in the presence of FGF 24.1 In the HUVECs treated with 50μM DBO for 24 h,the cell apoptotic rate increased from 21.3%to 68.5%and the content of integrinβ4 was also obviously increased(figure 26 and 27).4.2 DBO(50μM treatment for 12 h)significantly increased the level of ROS and NO in HUVECs(figure 28 and 30A).Enzyme activity assay showed that the activity of NADPH oxidase and iNOS obviously increased,but that of SOD and eNOS did not obviously changed(figure 29 and 30B).The data indicated that DBO elevated the level of ROS and NO though increasing the activity of NADPH oxidase and iNOS.When NADPH oxidase was inhibited by its specific inhibitor dibenziodolium chloride(DPI),DBO could not elevate the levels of ROS and NO in HUVECs(figure 31).The results indicated that NO metabolism might be related to ROS metabolism in the process of VEC apoptosis.5.The effects of DBO on angiogenesisHUVECs treated with 50μM DBO for 24 h could differentiate towards capillary-like tubes to most extent on matrigel.But,with the prolonged culture, the formation of capillary-like tubes was inhibited by DBO.At 72 h,almost all capillary-like tubes were disrupted in DBO treatment group(figure 32).When the filter discs were soaked with DBO(10-40 nmol),some allantoic vessels existed while many capillary vessels disappeared on incubation for 3 days(figure 33).The data indicated that DBO effectively inhibited angiogenesis.Conclusions:1.The data suggested that the morpholin-3-one derivatives were new apoptosis inducers.Morpholin-3-one derivative compound 3 was the most effective one.2.Integrinβ4 might be a key factor in regulating lung adenocarcinoma cell survival and autophagic cell death.3.Our data suggested that DBO might inhibit angiogenesis through inducing VEC apoptosis.